Method for expressing recombinant genes in bacteria in absence of antibiotic selection

ABSTRACT

The invention provides a method for the expression of recombinant molecules in bacterial hosts in a defined medium in the absence of antibiotic selection. The method uses an expression vector comprising a regulatable promoter by which the production of foreign proteins may be controlled during the growth phase of the culture, an origin of replication maintaining medium vector copy number and a transcriptional terminator.

This application is the national stage filing under 35 U.S.C. 371 ofPCT/GB93/01547, filed Jul. 22, 1993.

FIELD OF THE INVENTION

This invention relates to a method for the expression of recombinantantibody genes in bacterial host cells in a defined medium in theabsence of antibiotic selection.

DESCRIPTION OF BACKGROUND ART

The rapid developments in recombinant DNA techniques have resulted inthe identification and isolation of many novel genes, some of knownfunction and some of unknown function. Invariably there is a need toexpress the gene in a heterologous cell system in order to producematerial for structure-function studies, diagnostic reagents such asmonoclonal or polyclonal antibodies and material for in vivo activitytesting and therapy.

Several alternative systems for the expression of foreign genes havebeen developed including systems based upon mammalian cells, insectcells, fungal cells, bacterial cells and transgenic animals or plants.The choice of expression system for a given gene depends upon the likelyfeatures of the encoded protein, for example any post-translationalprotein modifications needed for biological activity, as well as theobjective of the study. Other important considerations for theinvestigator are the facilities available, time and cost involved ingenerating the amounts or recombinant protein required.

The most widely used and convenient system for the production of foreignproteins remains that based on the prokaryote Escherichia coli. Theadvantages of this system comprise the ease of gene manipulation, theavailability of reagents including gene expression vectors, the ease ofproducing quantities of protein (up to a gramme in simple shake-flaskculture), speed and the high adaptability of the system to express awide variety of proteins.

Expression of any foreign gene in E. coli begins with the insertion of acDNA copy of the gene into an expression vector. Many forms ofexpression vector are available. Such vectors usually comprise a plasmidorigin of DNA replication, an antibiotic selectable marker and apromoter and transcriptional terminator separated by a multi-cloningsite (expression cassette) and a DNA sequence encoding a ribosomebinding site. The method of transcriptional regulation varies betweenthe various promoters now available (ptac, λpL, T7). The ptac and T7expression based systems are controlled by the chemical inducer IPTG,whilst the λ promoters are controlled by a temperature switch.

A problem encountered with E. coli based expression systems is thedifficulty of producing material which is acceptable for therapeuticuse. The use of complex media, antibiotic selection and potentiallyhazardous inducers such as IPTG may potentially render products such asrecombinant antibody fragments produced by E. coli fermentationtechnology unacceptable to the regulatory authorities for clinicalapplications. Evidence demonstrating clearance of these agents from thefinal product must be provided in order to secure regulatory clearance.Clearance of these agents, and especially demonstrating such clearance,is expensive. It is therefore desirable that an expression system shouldavoid the three above-mentioned problems.

Avoidance of these problems is not straightforward. Plasmids, especiallyexpression vectors, place a metabolic load on the host cell which actsas a selective pressure favouring loss of the plasmid from the cell.Therefore, in order to reduce the possibility of plasmid loss, orrearrangement of the plasmid to delete the expression activity, it isapparent that the metabolic load placed on a cell by other sourcesshould be reduced to a minimum. Therefore, the use of complex mediawhich contain, in addition to essential amino acids and minerals, avariety of naturally-sourced vitamins, cofactors and the like whichalleviate the metabolic load on the cell is favoured.

The term "complex medium" is used herein according to its well-knownsignification in the art, that is to denote a medium the exactformulation and chemical composition of which has not been determined.Frequently, such media are at least partly derived from natural sources.For example, it is well known to include bovine serum preparationscontaining a variety of uncharacterised vitamins, growth factors and thelike in cell culture media. In contrast, a defined medium is a mediumwhich has been formulated from pure ingredients each of which is known,the medium therefore having a defined formula.

The use of a complex medium introduces the possibility of inclusion of avariety of agents derived from the natural source of certain mediumcomponents of which the regulatory authorities will require proof ofclearance. Switching to the use of defined media would facilitateobtaining regulatory approval because it can be stated with certaintythat the potentially harmful agents are absent from the cell culture.However, the efficiency of protein expression would also be reducedbecause the metabolic load on the cells in culture may be increased andplasmid expression systems may therefore be destabilised.

A manufacturer producing therapeutic products in cell culture istherefore faced with deciding which route will prove the mostcost-effective. In the commercial production systems of the prior art,which use relatively unstable plasmid systems, the equation has oftenfavoured the use of complex media and subsequent clearancedemonstration.

The situation is similar in respect of antibiotics. These are commonlyused to select against plasmid loss by inclusion of an antibioticresistance gene on the plasmid. In the absence of antibiotic selection,the increased metabolic load placed on the cell by the plasmid acts tofavour growth of plasmid free cells. Therefore, culture of transformedcells without antibiotic is not economic because, even though clearanceof the antibiotic does not need to be demonstrated, the potential lossof plasmid from the cells may greatly reduce the efficiency of theexpression system.

It should be borne in mind that it is not necessarily the cost ofclearing the suspect agent from the product which is high, but ratherthe cost of providing evidence of this clearance. Thus, for example,IPTG may clear by degradation from the medium, but the cost ofdemonstrating the clearance of it and potential contaminants anddegradation products thereof remains substantial.

It follows, therefore, that it would be desirable to be able to expressproteins in a bacterial system in a defined medium in the absence ofantibiotic selection and unacceptable inducers such as IPTG. However,due to the problems of plasmid instability, this aim has not beenachieved in commercial expression systems of the prior art.

For example, a recent report describing expression of antibody fragmentsin E. coli host cells employs the antibiotic tetracycline to preventplasmid loss from the cells (Carter et al. BioTechnology 10, 163-167,1992). Carter et al employ an inducible expression system to preventheterologous protein expression during host cell growth. Selectiveadvantage for growth of plasmid free cells potentially enhanced by theuse of defined medium is avoided by the use of tetracycline to selectagainst such cells.

However, the use of antibiotic is required both during growth, evidentlybecause the vector being used is not sufficiently stable, and during theexpression phase. Although this system has the advantage that no inducerneed be added, the requirement for antibiotic is not removed, asevidenced by the use of tetracycline in the expression system described.Therefore, the method of Carter et al will require the demonstration ofclearance of antibiotic from the final product. This is hypothesised tobe necessary because the expression vector used by Carter et al is notsufficiently stable to be cultured in the absence of antibiotic.

Antibodies and antibody fragments, especially recombinant or humanisedderivatives thereof, are a class of proteins which it would be extremelydesirable to be able to produce by recombinant DNA technology. Byhumanised antibodies, it is intended to refer to antibodies in which theconstant regions are derived from human immunoglobulins, while at leastthe complementarity determining regions (CDRs) of the variable domainsare derived from murine monoclonal immunoglobulins.

A number of improvements over natural immunoglobulins have beendocumented in the literature, which can only he put into practice byrecombinant DNA technology. For instance, the production of CDR-graftedantibodies having CDRs from murine antibodies coupled to human frameworkregions can only be undertaken using a recombinant expression system.Furthermore, such systems are extremely useful for the production ofantibody fragments which are not readily obtained by proteolyticcleavage, such as Fv fragments, and antibody fusions comprising aneffector or reporter molecule attached to the antigen binding molecule.

Recombinant antibody fragments whether they be entire antibodies, Fab,Fab', F(ab')₂ or Fv fragments, consist of heavy and light chain dimers.A recombinant expression system should therefore be capable ofexpressing both heavy and light chain genes in such a manner as torender the individual peptides capable of self-assembly into the finalproduct. This has been a stumbling block for recombinant antibodyproduction, and indeed attempts have been made to solve the problem. Anexample of this is the production of "single chain" Fv fragments,wherein the heavy and light chain polypeptides are physically joinedtogether by a flexible linker group. These molecules avoid the problemsof chain association between free heavy and light chain polypeptides.

This system is not necessary, however, for the production of antibodyfragments such as Fabs, which comprise heavy and light constant regionchains as well as heavy and light variable region chains. For suchapplications it is desirable to express heavy and light chainsseparately in the same cell.

In order to facilitate correct assembly of heavy and light chains ofantibody fragments, it is preferable to employ an expression system inwhich the chains are secreted into the culture medium rather thanprecipitated into the cell as inclusion bodies.

The use of E. coli signal sequences fused to polypeptides in order tofacilitate their secretion by E. coli is known. However, it is apparentin a number of cases that secretion of heterologous proteins is evenmore deleterious for E. coli than accumulation of such proteinsintracellularly. Accordingly, it has been found necessary to control theexpression of heavy and light chain genes in order to achieve high cellgrowth and plasmid stability during the growth phase of a bacterialculture.

SUMMARY OF THE INVENTION

The present invention solves the above problems by the provision of amethod for the expression of recombinant molecules such as antibodymolecules in bacterial hosts in a defined medium in the absence ofantibiotic selection. Furthermore, the method of the invention uses anexpression vector comprising a regulatable promoter by which theproduction of foreign proteins may be controlled during the growth phaseof the culture, an origin of replication maintaining medium vector copynumber and a transcriptional terminator. An example of such a plasmidsystem is described in International Patent Application No. WO 92/01059where the vector was used to express antibody fragments. In accordancewith the teaching of the art, the host cell transformed with the vectorwas cultured in the presence of antibiotics. We have now mostunexpectedly found that host cells transformed with vectors of this typemay, in fact, be cultured in the absence of antibiotic selection.

According to a first aspect the invention provides a method forproducing one or more heterologous protein(s) in a bacterial host cellcomprising culturing a bacterial host cell transformed with one or moreexpression vector(s) comprising one or more heterologous DNA sequencesunder the control of at least one regulatable promoter, an origin ofreplication maintaining medium vector copy number and a transcriptionalterminator characterised in that said host cell is cultured in a definedmedium in the absence of antibiotic selection.

The expression vectors for use according to the method of the inventionwill characteristically show 100% structural stability as determined byrunning restriction digests of plasmid preparations made from cellsamples taken throughout fermentation up to and including harvester; andwill characteristically show segregational stability as demonstratedby >80% of the cell population maintaining antibiotic selection markerin defined medium with no antibiotic selection at the point of inductionand product expression.

The regulatable promoter is a promoter which tightly repressesexpression of heterologous DNA during the growth phase of the cultureand from which expression preferably may be achieved without addition ofchemical inducers such as IPTG.

The expression vector preferably further comprises a gene encoding arepressor which acts on the regulatable promoter to prevent expressionof the heterologous DNA sequence(s).

A novel induction system for use with such regulatable promoters isdescribed in our copending International patent application filed oneven date herewith and derived from British patent application number9215550.6 filed on Jul. 22, 1992. The regulatable promoter is thereforepreferably repressed by a mature endogenous cellular repressor in adefined medium under conditions such that the inducible promoter isrepressed, and expression therefrom is induced by increasing themetabolic rate of the host cell thereby depleting the levels of themature endogenous cellular repressor. Preferably the increase inmetabolic rate is brought about by a switch in carbon source such asfrom glycerol to glucose.

In a preferred embodiment the invention provides a method for producinga heterologous protein in a bacterial host cell comprising culturing abacterial host cell transformed with an expression vector comprising aheterologous DNA sequence under the control of a regulatable promoter,an origin of replication maintaining medium vector copy number and atranscriptional terminator characterised in that said host cell iscultured in a defined medium in the absence of antibiotic selection.

The heterologous DNA sequences may code for any eukaryotic polypeptidesuch as for example a mammalian polypeptide such as an enzyme e.g.chymosin or gastric lipase; an enzyme inhibitor e.g. TIMP; a hormonee.g. growth hormone; a lymphokine e.g. an interferon or interleukin; aplasminogen activator e.g. tPA.

The heterologous DNA sequence(s) will preferably each be fused to a DNAsequence encoding a secretion sequence said secretion sequence beingunder the control of a regulatable promoter.

The heterologous DNA sequence(s) will preferably encode antibodymolecules and fragments thereof and may therefore be gene(s) coding forall or part of an antibody heavy chain and/or light chain.

The antibody fragments may comprise natural antibody fragments, chimericantibody fragments (the variable domains derived from one species andclass of antibody and remaining Ig sequences derived from anotherspecies or class of Ig), altered antibody fragments (variable Ig domainsplus an additional polypeptide sequence having a different, non Igfunction, such as an enzyme or toxin), humanised antibody fragments andengineered antibody fragments (wherein the Ig amino acid sequence hasbeen altered from the natural sequence, e.g. by site-directedmutagenesis, with a view to altering a characteristic of the molecule,e.g. antigen binding specificity or affinity, for example as describedin Roberts et al., Nature, 328, 731-734, 1987). The antibody fragmentsmay comprise suitable combinations of the above types of antibodyfragment.

Preferably the antibody molecule is a humanised antibody moleculecoupling at least the CDRs of a non-human antibody attached to theframework of a human antibody.

Preferably, the antibody molecule is an antibody fragment. For example,the antibody molecule may be a Fab, Fab', (Fab')₂ or Fv fragment.Advantageously, it is an Fab' fragment.

The antibody fragments may have any desired antigen specificity. Forexample, the antibody fragments may have specificity for a cell-specificantigen, such as a tumour antigen, T cell marker, etc. Particularlypreferred are antibody fragments which have specificity fortumour-associated antigens such as CEA and TAG72. Chimeric A5B7antibodies and antibody fragments are described in our copendingInternational patent application WO92/01059. Also preferred areantibodies having specificity to the epitope recognised by murinemonoclonal antibody A33 as described in our copending British patentapplication number 9225853.2 filed Dec. 10, 1992. Particularly preferredare humanised and chimeric forms of A33, and especially preferred areFab' fragments thereof.

The antibodies may be site-specific antibodies such as tumour-specificor cell surface-specific antibodies, suitable for use in in vivo therapyor diagnosis, e.g. tumour imaging. Examples of cell surface-specificantibodies are anti-T cell antibodies, such as anti-CD3, and CD4 andadhesion molecules, such as CR3, ICAM and ELAM. The antibodies may havespecificity for interleukins (including lymphokines, growth factors andstimulating factors), hormones and other biologically active compounds,and receptors for any of these. For example, the antibodies may havespecificity for any of the following: Interferons α, β, γ or δ, IL1,IL2, IL3 or IL4, etc., TNF, GCSF, GMCSF, EPO, hGH, or insulin, etc.

Preferably, the copy number of the expression vector is between 6 and50. Advantageously, it is between 10 and 20 and most preferably it is15.

According to a preferred embodiment of the first aspect of theinvention, therefore, there is provided a method for producing antibodymolecules or fragments thereof comprising culturing a bacterial hostcell transformed with an expression vector comprising a heterologous DNAsequence coding for all or part of a heavy chain and a light chain underthe control of a regulatable promoter, an origin of replicationmaintaining medium copy number and a transcriptional terminatorcharacterised in that said host cell is cultured in a defined medium inthe absence of antibiotic selection.

The heterologous DNA sequence coding for all or part of a heavy and alight chain will conveniently be under the control of one regulatablepromoter. In some instances it may, however, be possible to place eachof the heavy and light chain genes under the control of a separateregulatable promoter, which may be the same or different. Heavy andlight chains may also be encoded on separate vectors which areco-transformed and expressed as described in UK Patent No. 2137631B.

Preferably the heavy chain and light chain genes are each fused to a DNAsequence encoding a secretion sequence which may be under the control ofthe regulatable promoter.

The use of an origin of replication which avoids the single-strandedstate during DNA replication has been found to be particularlyadvantageous in stabilising the plasmid under conditions of highmetabolic load. It is hypothesised that, although the metabolic loadinduced by the plasmid itself is not lowered, the avoidance of thesingle-stranded state allows the plasmid to replicate more safely,reducing the probability of attack by cellular nucleases or otherdegrading agents.

Preferably, the medium copy-number origin of replication allows plasmidreplication without passing through a single stranded DNA phase.

Preferably, the origin of replication is derived from pSC101 Hashimotoet al (Gene 16 227-235 (1981)) which is a medium copy number plasmidwhich does not replicate through a single-stranded phase. Preferably,the regulatable promoter/inducer system selected will be one regulationof which is by means of a a clinically acceptable inducer of whichclearance demonstration will not be required by the regulatoryauthorities.

Preferably, the regulatable promoter is the tac promoter and therepressor is the lacI^(Q) repressor. The advantage of using the lacI^(Q)repressor is that lactose may be used to induce expression from the tacpromoter by inactivating the repressor. This is desirable since lactoseis likely to be a clinically acceptable inducing agent.

An antibiotic resistance gene is included in order to allow theapplication of selective pressure during the growth of the bacteria inorder to select for plasmid retention. The inclusion of an antibioticresistance gene is essential in order to allow for selection duringconstruction of the plasmid. During the expression phase, no antibioticis added, since the use of antibiotics is undesirable from a clinicaland regulatory point of view.

A further advantage of this system is that whereas it is, at present,acceptable to demonstrate clearance of certain substances in order tocomply with regulatory requirements, in future it may become stronglydiscouraged to use such substances at all. Therefore an expressionsystem capable of operating in antibiotic-free defined media may becomeessential for the production of therapeutic material.

According to a further aspect of the invention, the vector for use inthe method of the invention preferably comprises heavy chain and lightchain genes arranged with the light chain gene located closer to thepromoter such that it is transcribed first. It has been observed that byplacing the light chain gene closer to the promoter, in such a mannerthat it is translationally coupled to the gene which the promoter isdirectly coupled to, and placing the heavy chain gene downstream fromthe light chain gene in such a manner that it does not benefit fromtranslational coupling, both cell viability and efficiency of antibodysecretion are enhanced.

In order to effect translational coupling, the natural coding sequenceof the bacterial gene whose promoter is being used in the expressionvector is altered in order to introduce a stop codon just before thebeginning of the sequence of the inserted heterologous gene. It ishypothesized that this causes ribosomes, which are efficiently assembledon the mRNA of the bacterial coding sequence, to become disengaged inthe close proximity of the translational start site of the heterologousmRNA. This favours the reassembly of the ribosomes on the heterologousmRNA, thus increasing the level of expression.

It is postulated that expression of an excess of heavy chains isdeleterious to the host cell. However, arranging the light chain genesuch that expression of light chain is favoured ensures an excess oflight chains in the cell, thus avoiding the problems associated withexcess heavy chain production. The arrangement of cistrons described isdesigned to favour the expression of an excess of light chains.

In a preferred embodiment of the vector for use in the method of theinvention, the vector includes a secretion sequence to effect secretionof antibody heavy and/or light chains. The use of E. coli ompA secretionsequences is especially preferred Preferably, the ompA translationinitiation signals are also included.

Advantageously, the ompA-antibody light chain fusion is translationallycoupled to the lacZ peptide translated from the tac promoter. This mayrequire the alteration of the ompA translation initiation sequence tointroduce a stop codon.

Preferably, secretion of the antibody chains from the host cells iscarried out at 30° C. It has been found that more protein is secreted at30° C. than at 37° C., presumably because at the lower temperature theprotein is more likely to adopt a translocation-competent state beforesecretion, or to remain soluble after secretion.

Preferably, the bacterial host cell is a gram-negative bacterial hostcell. Preferably, the host cell is an E. coli cell.

The expression vectors are preferably based on vectors disclosed inInternational patent application WO92/01059, and are preferably pACtacderivatives.

Examples of chemically defined medium are provided in Pirt S J (1975)"Principles of Microbe and Cell Cultivation", Blackwell ScientificPublications. Further examples of chemically defined medium are providedin the examples included herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is now described by way of example only with reference tothe following figures, in which:

FIG. 1 is a diagrammatic representation of the vectors of the inventionand intermediates used in their preparation;

FIG. 2 is a photograph of an agarose gel electrophoresis experiment ofplasmid preparations derived from bacterial cultures transfected withplasmids comprising heavy and light chain genes in the order cLc-cFd'and cFd'-cLc;

FIG. 3 is an immunoblot of cultures of bacteria transformed with vectorsaccording to the invention;

FIG. 4 is a photograph of a western blot which compares the yields ofantibody product from dual origin vectors transfected with cLc-cFd' andcFd'-cLc gene constructs;

FIG. 5 is an immunoblot comparing the performance of two differentstrains of bacteria transformed with a vector according to theinvention;

FIG. 6 is an immunoblot showing the performance of host cellstransformed with a vector according to the invention in a definedmedium;

FIG. 7 is a graph of CEA binding assays on crude culture fermentationsamples from the defined medium cultures;

FIG. 8 shows the production of antibody fragments when cultures areinduced by lactose;

FIG. 9 shows the growth rate of host cells transformed with a vectoraccording to the invention in a defined growth medium with induction ofgene expression by lactose feeding;

FIG. 10 shows the accumulation profile of Fab' protein produced in theexperiment of FIG. 9; and

FIG. 11 is a western blot of the extract from the cells grown in theexperiment of FIGS. 9 and 10.

DETAILED DESCRIPTION OF THE INVENTION Experimental Construction of Heavyand Light-Chain Fusions

Plasmid pQ9kan was constructed by reorganisation of the commerciallyavailable vector pTTQ9 (Amersham International plc). pTTQ9 was digestedwith Sca I, which cuts within the ampicillin resistance gene. A 1.3 kbHinc II fragment encoding the kanamycin resistance gene was isolatedfrom pUC4K (Pharmacia LKB) and ligated into Sca I cut pTTQ9. Clones withthe kanamycin resistance gene inserted in the correct orientation wereisolated, and are referred to as pQ9Kan.

A5B7 heavy and light chain sequences cLc (chimeric light chain) and cFd'(chimeric Fd' heavy chain fragment) were isolated as described inInternational patent application WO 92/01059. These were fused to the E.coli secretion sequence ompA and termed ompA-cLc and ompA-cFd', asdescribed in International patent application WO 92101059. The sequenceswere inserted into pSK (Stratagene) as described in WO 92/01059 toproduce pSKompA-cLc and pSKompA-cFd'.

Insertion of ompA-cLc and ompA-cFd' into pQ9Kan was carried out bydigesting pQ9Kan with Sal I and Eco RI and inserting the requiredsequence isolated from pSKompA-cLc or pSKompA-cFd' as required, as XhoI-Eco RI fragments, to give pQ9Kan-cLc and pQ9Kan-cFd'. To formpQ9Kan-cLc-cFd', pQ9Kan-cLc was digested with Eco RI. The protruding 5'tail was filled in with T4 DNA polymerase to form a blunt end. TheompA-cFd' gene was isolated as an Xho I-Eco RI fragment frompSKompA-cFd', and filled in similarly. Ligation of the two filled inproducts and subsequent selection for the desired orientation ofinsertion yielded pQ9Kan-cLc-cFd'.

pQ9Kan-cFd'-cLc was constructed in an analogous fashion from pQ9Kan-cFd'Eco RI (filled) and the ompA-cLc gene digested with Eco RI and Xho I,and filled in.

Expression of Chimeric A5B7 Fab' in pQ9kan

Expression and stability studies were performed with plasmidspQ9Kan-cLc-cFd' and pQ9Kan-cFd'-cLc in E. coli strains XL1 Blue andW3110, in both shake flasks and 2 l fermentors. A map of the former isshown in FIG. 1, and the latter is identical except for the relativepositions of ompA-cLc and ompA-cFd' fusions with respect to the tacpromoter. In shake flask experiments both plasmids displayed serioussegregational instability in XL1Blue, even in non-inducing conditionswith kanamycin present. In W3110 pQ9Kan-cFd'-cLc appeared somewhat lesssegregationally unstable but nevertheless showed serious and progressiveplasmid loss (see FIG. 2). FIG. 2 is an Agarose gel showing preparationsof pQ9Kan-cLc-cFd' and pQ9Kan-cFd'-cLc plasmids. Lane 1: λ-Hind IIImarkers; lanes 2 to 11: plasmid preparations from strain W3110transformed with pQ9Kan-cLc-cFd' (A5B7); lanes 12 to 16: plasmidpreparations from strain W3110 transformed with pQ9Kan-cFd'-cLc (A5B7).

In lanes 2 to 11, the intact plasmid can be seen just above the 4.4 Kbmarker and no degradation products are apparent. In lanes 12 to 16, onthe other hand, degradation products may be seen at about 2.1 and 4 Kb.

W3110/pQ9Kan-cLc-cFd' proved the most stable strain/plasmid combination,an approximately constant proportion (50%) of the cells retaining theplasmid in non-inducing conditions. This strain, however, consistentlyfailed to grow in fermentors, and unlike the other three strains alsoconsistently failed to grow beyond OD600 of 10 in shake flask cultures.In structural stability studies in fermentors on the other three strainspQ9Kan-cFd'-cLc was observed to suffer deletions in both strains innon-inducing conditions, while pQ9Kan-cLc-cFd' appeared structurallystable in XL1Blue.

It was concluded that the promoter-cLc-cFd' order gives betterstructural stability than the promoter-cFd'-cLc order, and that only theW3110/pQ9Kan-cLc-cFd' combination was sufficiently stable for expressionexperiments to the meaningful. When shake flask cultures of this strainwere induced at an OD600 of 5, Western immunoblotting revealed thepresence of two immunoreacting bands roughly of the size expected ofFab' fragments in cell extracts but not in culture supernatants.Optimisation of induction times and feeding of nutrients after inductionimproved the yield of this material in cell extracts and led to itsappearance in culture supernatants. Rough estimates of yield bycomparison on immunoblots with measured amounts of chimeric Fab' made inCHO cells suggested a yield of about 5 mgs/l in these culturesupematants. Because this strain was unable to grow in fermentorsexpression constructs were made and tested using other vectors.

Expression of Chimeric A5B7 Fab' in pACtac

pACtac is a medium copy number (approx 15/cell) relative of pQ9kan. Itwas constructed by replacing the kanamycin selectable marker and pUCreplication functions of the latter with those of pACYC184 (see FIG. 1)as described in International patent application WO 92/01059. Theplasmids pSKompA-cLc and pSKompA-cFd' contain the A5B7 chimeric lightand heavy chains respectively fused precisely to the ompA signalsequence as described above, inserted into pSK.

The ompA-cic and ompA-cFd' fusions were isolated as Xhol-Sma1 fragmentsand from pSKompA-cLc and pSKompA-cFd' respectively and cloned into theSal1-Pvull gap of pSP73 (Promega Corp.) to give plasmids pMMR026 andpMRR027 respectively, this to allow subsequent manipulation as EcoR1fragments. The EcoR1 fragment of pMRR027 carrying the ompA-cFd' fusionwas then cloned into pMRR024 (see WO 92/01059), partially cleaved withEcoR1 to give pMRR028 (see FIG. 1), which thus carried the Fab' genes inthe order promoter-cLc-cFd'. pMRR028 was transformed into strains W3110and XL1Blue. Expression studies were carried out first on XL1Blue(pMRR028) to provide a direct comparison with results for the twoplasmid system.

Shake flask cultures of XL1Blue (pMRR028) in YEGLY medium pluschloramphenicol were induced at an OD600 of 10 with 200 μM IPTG. Westernimmunoblotting of culture supernatants along with a series of measuredamounts of purified chimeric A5B7 Fab's suggested a yield in excess of30 mg/l. As FIG. 3 shows no Fab' was detected in cell extracts ofuninduced cultures, suggesting extremely tight regulation of expressionfor this strain. Structural and segregational stability tests showed nosign of instability through the six or seven generations in YEGLY mediumplus chloramphenicol examined in these experiments for either induced oruninduced cultures.

FIG. 3 is an Immunoblot of samples from shake flask cultures of strainXL1 Blue (pMRR028).

lane 1 shows a supernatant sample from a fermentation of XL1 Blue(pQ9kan-cLc, pMRR025)

lanes 2, 4 and 5 supernatant samples from induced cultures of XL1 Blue(pMRR028)

lanes 3 and 6 supernatant samples from uninduced cultures of XL1 Blue(pMRR028)

lanes 7, 9 and 10 cell extracts from induced cultures of XL1 Blue(pMRR028)

lane 8 cell extract from an uninduced culture of XL1 Blue (pMRR028)

1.5 l batch fermentations were performed using the same medium andinduction conditions for both XL1Blue (pMRR028) and W3110 (pMRR028).Immunoblotting suggested roughly equal yields for both strains.

FIG. 4 is a Western blot of culture supematants and cell extracts fromcell lines transformed with DUOV vectors transformed with cLc-cFd' andcFd'-cLc genes.

lane 12 culture supernatant of XL1B transfected with pMRR032 (cLc-cFd');

lanes 13 and 14 culture supernatants of XL1B transfected with pMRR033(cFd'-cLc).

lane 15 cell extract of XL1B transfected with pMRR032

lanes 16 and 17 cell extract of XL1B transfected with pMRR033. ThecLc-cFd' gene order can be seen to give much higher yields of product.

As shown in FIG. 4, however, one very significant difference wasobserved between the Fab' products appearing in the culture supernatantsof XL1Blue and W3110--the latter appeared to be secreting only thenormal sized light chain. This immunoblot revealed that the slowermigrating form of the light chain is, however, present in the W3110 cellextracts. This suggests selective release of the normal sized lightchain by this strain, rather than selective synthesis.

Expression and Fermentation Development Towards a Clinically AcceptableProcess

In order to develop a clinically-compatible fermentation process lactoseinstead of IPTG was used for induction of pMMR028 in W3110. Thisapproach involves the use of defined medium without antibioticselection, in the production fermentor.

Subculturing experiments in complex medium were performed for W3110(pMRR028) to assess the segregational and structural stability of theplasmid. No plasmid loss or deletions were observed in this medium overthe six to seven generations which are required in the productionfermentation (1000 l scale)--see FIG. 5.

FIG. 5 is an immunoblot of samples from fermentations of XL1 Blue(pMRR028) and W3110 (pMRR028).

lane 1 molecular weight markers

lanes 2-10 culture supernatant samples 1-9 from XL1 Blue (pMRR028).

lanes 11 and 12 cell extracts from XL1 Blue (pMRR028) samples 6 and 9

lanes 13-17 culture supernatant samples 1, 3, 5, 7 and 9 from W3110(pMRR028)

lanes 18 and 19 cell extracts from W3110 samples 6 and 9.

Samples were taken approximately hourly from t=10 hours, with inductionsat time of sample 3 for XL1 Blue (pMRR028) and sample 1 for W3110(pMRR028).

Controlled exponential phase cultures of this strain have been grown indefined minimal salts medium to a cell density (OD600 of >30)approaching that which is required for high yielding productionfermentations. Such cultures were successfully induced with IPTG (seeFIG. 6) and shown to give yields similar to those observed for YEGLYmedium.

FIG. 6 is an immunoblot of samples from fermentations of W3110 (pMRR028)in defined medium.

lanes 1 and 3 cell extracts

lanes 2, 4 and 5 culture supernatant samples

CEA binding assays were performed on crude supernatant samples fromthese fermentations, with the results shown in FIG. 7. As this Figureindicates the supematants contain material active in antigen binding.

Induction of Fab' expression for W3110 (pMRR028) growing in YEGLY mediumin a 1.5 l fermentor has also been achieved by feeding lactose ratherthan adding IPTG batchwise. A Western immunoblot of cell extract andsupernatant samples from this fermentation is shown in FIG. 8, andsuggests relatively low levels of Fab' expression and secretion.Interestingly the cell-bound immunoreacting material shows a muchgreater proportion of the normal sized light chain than observed withIPTG. Induction appeared to occur late in the fermentation, some 8-10hours after the start of lactose feeding.

FIG. 8 is an immunoblot of samples from a fermentation of W3110(pMRR028) in YEGLY medium, with induction by lactose feeding.

lane 1 chimeric A5B7 Fab' standard purified from CHO cells

lanes 2 and 3 cell extracts after induction

lanes 4 and 5 culture supernatant samples after induction

The OUR profile of this fermentation showed an isolated peak around thistime which may have represented a switch to utilisation of lactose uponexhaustion of preferred carbon sources, with consequent induction byallolactose.

These findings were supported by a subsequent experiment whichdemonstrated actual expression of A5B7 Fab' in a defined,antibiotic-free medium under lactose induction. Fermentations wereperformed with strain W3110 carrying pMRR028 in defined medium usingglucose as the initial carbon source prior to switching to lactose fedat a rate suitable for achieving induction and for supporting furthergrowth. The growth curve from one such fermentation is given in FIG. 9,which demonstrates diauxic growth. The first growth phase ceased at thepoint of glucose exhaustion, which occurred at an OD600 of about 30. Thespecific growth rate in the second phase was lower but continued up tothe point of harvest. This lower specific growth rate should allowgreater cell concentrations to be kept oxygen sufficient. Accumulationof product, as defined by both Coomassie Blue binding for proteinestimation and CEA-binding, is shown in FIG. 10. Accumulation of activeFab' in the medium continued up to the point of harvest. Westernimmunoblots revealed the same four principal species seen in the culturesupernatant for the dual origin system.

Reduced cell extracts of W3110 (pMRR028) showed a single band onimmunoblots of SDS-PAGE. This species is 27 kD molecular weight, is themajor cell-associated protein in induced cells, is not present inuninduced cells and co-migrates with active Fab' made in mammalian cells(see FIG. 11). FIG. 11 is an SDS-PAGE of E. coli W3110 (pMRR028) cellextracts.

Cell extracts were electrophoresed on a 12% reducing gel.

lane 4 before induction;

lanes 2 and 5 4 hours after induction;

lanes 3 and 6 18 hours after induction.

1--cFab' standard from CHO cells. Bands A and B are the two proteinsappearing only after induction. Only band B is released into the medium.

It proved possible to observe CEA binding activity in cell lysates froma wide range of fermentations, with titres up to 250 mg Fab'/L oflysate.

EXAMPLE A Methods for the Expression of A33 Grafted Fab' in E. coliUsing the pAC tac Expression Vector

Humanised A33 Fab' may be constructed and expressed in E. coli asdescribed in British patent application number 9225853.2 filed Dec. 10,1992 and initial British patent application filed on even date herewith(ref. PA 345).

1. Storage of Strains

2. Revival of Cultures and Inoculum Preparation

3. Media

4. Fermentor Culture and Induction Procedures

5. Harvesting

6. Periplasmic Extraction Procedures.

1. STORAGE OF STRAINS

A single colony from a freshly transformed plate was streaked out on anLA chloramphenicol plate. A single colony from this plate was used toinoculate a 250 ml Erlenmeyer flask containing 40 ml LBmedium+chloramphenicol. This flask was incubated at 30° C. and 250 RPMin an orbital incubator until the culture reached an optical density (OD600 nm) of 2 (mid exponential growth phase) taking approximately Bh toreach this point. Aliquots (750 μl) of this culture were mixed with 250μl sterile glycerol solution (50% v/v in H₂ O) in a 2 ml sterile ampoule(Sterilin). These glycerol stocks were stored at -70° C. withoutcontrolling freezing rate.

Lyophilised stocks were prepared from a similar culture to thatdescribed above (from the same original transformant). Sterile sucrosesolution rather than glycerol was added as a cryoprotectant (afterincubation) to a final concentration of 10% (w/v).

2. REVIVAL OF STRAINS AND INOCULUM PREPARATION

Inocula for all experiments were prepared from frozen glycerol stocks inLB medium containing Cm or Amp as appropriate, the seeding density wasusually 300 μl glycerol stock per liter LB. Inoculum cultures, grown inErlenmeyer flasks (1L containing 200 ml medium) incubated at 30° C. and250 RPM in an orbital shaker were used when an OD 600 nm of 3 had beenattained (normally 12-16 h). Fermentors and shake flasks were seededwith 5-10% volumes of inoculum.

    ______________________________________    3.  MEDIA    ______________________________________    3.1  LA: Luria Agar        LA Cm: LA + chloramphenicol 25 μg/ml    ______________________________________    3.2 LB: Luria Broth        LB Cm: LB + chloramphenicol 25 μg/ml    ______________________________________    3.3 SM6B        Component                 g/L    ______________________________________    (NH.sub.4).sub.2 SO.sub.4 5.0    NaH.sub.2 PO.sub.4        6.24    KCl                       3.87    MgSO.sub.4.1H.sub.2 O     0.56    Citrate                   4.0    SM6A Trace Element solution                               10 ml/L    Antifoam (Mazu DF843 10% in H.sub.2 O)                              1.0 ml/L    Made up to 0.2 L with deionised water (5x solution)    ______________________________________    SM6B Trace element solution                              g/L 100x    Component                 stock solution    ______________________________________    Citrate                   100.0    CaCl.sub.2.6H.sub.2 O     5.0    ZnSO.sub.4.4H.sub.2 O     2.0    MnSO.sub.4.4H.sub.2 O     2.0    CuSO.sub.4.5H.sub.2 O     0.5    CoSO.sub.4.6H.sub.2 O     0.4    FeCl.sub.3.6H.sub.2 O     9.67    H.sub.3 BO.sub.3          0.03    NaMoO.sub.4               0.02    ______________________________________

Made up to 1 l with deionised water. Components were added in the ordershown and were allowed to dissolve completely prior to the addition ofthe next salt.

Medium SM6B was kept as a 5× solution, the concentrated salts solutionwas added to the fermentor to the correct concentration for the culturevolume up to the point of induction (i.e. sterilisedvolume+inoculum+glucose feeds). Subsequent salt requirements arisingfrom the increase in volume brought about by feeding lactose solutionwere supplied at the time of lactose feeding. The fermentor was broughtup to the correct volume pre sterilisation with deionised water.

Defined media were brought to pH 6.95 using 3.6M NH₄ OH afterautoclaving in situ at 121° C. for 20 min. After sterilisation of thesalts solution, glucose was added to the fermentor as a 50% (w/v)solution to a final concentration of 20 g/L.

Glucose and lactose were autoclaved separately as 50% solution (w/v) inH₂ O and added to cultures as described in the fermentation methodssection. Prior to autoclaving, conc H₂ SO₄ (100 μl per liter) was addedto glucose solutions.

Casamino acids (Difco, 200 g/l) solution in H₂ O sterilised byautoclaving) were added to the fermentor at the start of lactose feedingto give a final accumulated supply of 20 g/L (final fermentor volume).

4. FERMENTATION CONDITIONS

Fermentations were made in medium SM6B, glucose was used as the initialcarbon and energy source for all fermentations and was added aftermedium sterilisation to a concentration of 20 g/l. Culture pH wasbrought to and maintained at 6.95 by the addition of 3.6M NH₄ OH or 2MH₂ SO₄. Dissolved oxygen tension (DOT) was maintained above 10% airsaturation by control of agitator speed (between 250 and 1000 RPM for 2and 15L fermentations and between 150 and 650 RPM for 150Lfermentations) culture temperature was maintained at 30° C. throughoutthe fermentation. Cultures were aerated at 0.75-1.5 v/v/min. During thelater stages of 150L fermentations the vessel was pressurised to 0.4 barto maintain the DOT above 10%. Oxygen utilisation rates (OUR) and carbondioxide evolution rates (CER) were determined from exhaust gas analysisvalues carried out by mass spectrometry. OUR reached maximum values ofapprox. 150 mmol/l/h in the fermentations described.

Lactose inductions; Induction of product expression was initiated byswitching the carbon source to lactose from glucose. Glucose was fed tosupport the culture to an OD of approximately 40 (an accumulativeaddition of 40 g/l). Lactose feeding was started at an OD ofapproximately 35, as with glucose, lactose was fed as individual shotsof 60% lactose to a concentration of up to 60 g/l culture when requiredor as a predetermined exponential feed program.

Cultures induced by lactose feeding were harvested 24 h after the switchfrom glucose to lactose utilisation.

Where casamino acids were added, these additions were made or started,at the start of lactose feeding.

5. HARVESTING

Fermentors were harvested 24 h after the switch to lactose utilisation.2L fermentations were clarified by centrifugation 4200 RPM max 250 mm.15 and 150L fermentations were clarified by tangential flow filtration(TFF) using a Millipore prostack system with durapore 0.65 μm membranesand a retentate flow rate of approx. 10 L/min/channel. Clarification offermentation broths by TFF was superior to scaleable centrifugationprocesses.

6. PERIPLASMIC EXTRACTION PROCEDURES

Product was released from the periplasm by incubating culture pellets orconcentrated cell suspensions harvested by centrifugation or TFFrespectively. Harvested cells were washed in Tris HCl buffer 100 mM pH7.4 and then incubated in Tris buffer 100 mM pH 7.4 containing 10 mMEDTA. Incubations were made at 40° C. for 4H. Repeated incubations ofthe cells produced further material.

EXAMPLE B Methods for the Expression of Antibody Fragments in E. coliUsing the Dual Origin and pAC tac Expression Vectors

1. Storage of Strains

2. Revival of Cultures and Inoculum Preparation

3. Media

4. Shake Flask Culture and Induction Procedures

4.1 Host strain W3110 with pAC tac vector

4.2 Host strain W3110 with dual origin vector

5. Fermentor Culture and Induction Procedures

5.1 Host strain W3110 with pAC tac vector

5.2 Host strain W3110 with dual origin vector

6. Periplasmic Extraction Procedures

1. STORAGE OF STRAINS

A single colony from a freshly transformed plate was streaked out on anLA plate containing the appropriate antibiotic selection. A singlecolony from this plate was used to inoculate a 250 ml Erlenmeyer flaskcontaining 40 ml LB medium+appropriate antibiotic selection (dual originvector: ampicillin, pAC tac vector: chloramphenicol). This flask wasincubated at 30° C. and 250 RPM in an orbital incubator until theculture reached an optical density (OD 600 nm) of 2 (mid exponentialgrowth phase) taking approximately 8 h to reach this point.

Aliquots (750 μl) of this culture were mixed with 250 μl sterileglycerol solution (50% v/v in H₂ O) in a 2 ml sterile ampoule (Sterlin).These glycerol stocks were stored at -70° C. without controllingfreezing rate.

2. REVIVAL OF STRAINS AND INOCULUM PREPARATION

Inocula for all experiments were prepared from frozen glycerol stocks inLB medium containing Cm or Amp as appropriate, the seeding density wasusually 300 μl glycerol stock per liter LB. Inoculum cultures, grown inErlenmeyer flasks (1L containing 200 ml medium) incubated at 30° C. and250 RPM in an orbital shaker were used when an OD 600 nm of 3 had beenattained (normally 12-16 h). Fermentors and shake flasks were seededwith 5-10% volumes of inoculum.

    ______________________________________    3.  MEDIA    ______________________________________    3.1 LA: Luria Agar        LA Cm: LA + chloramphenicol 25 μg/ml        LA Amp: LA + ampicillin 25 μg/ml    ______________________________________    3.2 LB: Luria Broth        LB Cm and LB Amp both 25 μg/ml    ______________________________________    3.3 YEGLY        Component                    g/L    ______________________________________    Glycerol                     20.0    (NH.sub.4).sub.2 SO.sub.4    7.0    NaH.sub.2 PO.sub.4.2H.sub.2 O                                 6.24    Yeast extract (Difco)        40.0    SM6 trace elements           10 ml/L    Antifoam solution (1) % mazu DF843)                                  1 ml/L    This formulation was made up to 1 L with deionised water.    ______________________________________    3.4 SM6        Component                    g/L    ______________________________________    (NH.sub.4).sub.2 SO.sub.4    5    NaH.sub.2 PO.sub.4           6.24    Trace element solution (SM6) 10 ml/L    Antifoam solution (10% mazu DF843)                                  1 ml/L    ______________________________________

This formulation was made up to 0.96L with deionised water. Where thecarbon source used was glycerol this was added to a concentration of 20g/L prior to autoclaving. Where glucose and or lactose were used thesewere added post sterilisation as 50% solutions (sterilised byautoclaving) to final concentrations of 20 g/L.

    ______________________________________    SM6 Trace element solution    Component        g/L 100x stock solution    ______________________________________    NaOH             15.0    EDTA             60.0    MgSO.sub.4.7H.sub.2 O                     20.0    CaCl.sub.2.6H.sub.2 O                     5.0    ZnSO.sub.4.4H.sub.2 O                     2.0    MnSO.sub.4.4H.sub.2 O                     2.0    CuSO.sub.4.5H.sub.2 O                     0.5    CoCl.sub.2.6H.sub.2 O                     0.095    FeSO.sub.4.7H.sub.2 O                     10.0    H.sub.3 BO.sub.3 0.031    Na.sub.2 MoO.sub.4                     0.002    ______________________________________

Each component was dissolved individually in deionised water and addedto the bulk solution in the sequence shown to a final volume of 1L.

    ______________________________________    3.5    SM6A           Component           g/L    ______________________________________    (NH.sub.4)SO.sub.4     5.0    NaH.sub.2 PO.sub.4     6.24    KCl                    3.87    MgSO.sub.4.1H.sub.2 O  0.56    Citrate                4.0    SM6A Trace Element solution                            10 ml/L    Antifoam (Mazu DF843 10% in H.sub.2 O)                           1.0 ml/L    ______________________________________

Made up to 0.95L with deionised water.

    ______________________________________    SM6A Trace element solution    Component        g/L 100x stock solution    ______________________________________    Citrate          100.0    CaCl.sub.2.6H.sub.2 O                     5.0    ZnSO.sub.4.4H.sub.2 O                     2.0    MnSO.sub.4.4H.sub.2 O                     2.0    CuSO.sub.4.5H.sub.2 O                     0.5    CoSO.sub.4.6H.sub.2 O                     0.4    FeCl.sub.3.6H.sub.2 O                     9.67    H.sub.3 BO.sub.3 0.03    NaMoO.sub.4      0.02    KCl              74.5    ______________________________________

Made up to 1 l with deionised water. Components were added in the ordershown and were allowed to dissolve completely prior to the addition ofthe next salt.

Defined media were brought to pH 6.95 using 3.6M NH₄ OH afterautoclaving.

Carbon sources for defined media were as described in the fermentationmethods section.

All media were sterilised by autoclaving at 121° C. for 20 min.

Glucose and Lactose were autoclaved separately as 50% solutions (w/v) inH₂ O and added to cultures as described in the fermentation methodssection. Prior to autoclaving, conc H₂ SO₄ (100 μl per liter) was addedto glucose solutions.

Glycerol for feeding during fermentations was autoclaved neat or as a50% w/v solution in H₂ O.

Casamino acids (Difco, 200 g/l solution in H₂ O sterilised byautoclaving) were added to give a final concentration of 20 g/L wheredescribed.

4. SHAKE FLASK CULTURE AND INDUCTION PROCEDURES

4.1 Host Strain W3110 with pAC tac Expression Vector

Shake flask cultures were made in 250 ml Erlenmeyer baffled flaskscontaining 40 ml YEGLY medium seeded with 4 ml inoculum prepared asdescribed in section 2. Cultures were incubated at 30° C. and 250 RPM inan orbital incubator. Induction of product expression was obtained at OD600 nm=5 by adding a 40 μl aliquot of IPTG (200 mM, freshly preparedaqueous solution sterilised by filtration). Cultures induced at an OD600 of 2.5 produced higher yields for certain fragments than thoseinduced at 5. Addition of IPTG to cultures which had reached OD's of 6or greater and had moved into the decline phase of growth did not induceproduct expression.

Culture supernatants were harvested by centrifugation 12 h postinduction with IPTG.

4.2 Host Strain W3110 with the Dual Origin Expression Vector

Cultures were grown as described in Section 4.1. Induction of productexpression was achieved by transferring flasks to an orbital incubatorpre equilibrated at 40° C. when cultures had reached an OD of 5.

Culture supernatants were harvested by centrifugation 12 h postinduction by temperature switching.

5. FERMENTOR CULTURE AND INDUCTION PROCEDURES

5.1 Host Strain W3110 with pAC tac Expression Vector

5.1.1 Complex medium fermentations

Fermentations were made in YEGLY medium inoculated at a seeding densityof 5% with the culture described in section 1. The culture pH wascontrolled at 7.0+/-0.05 by addition of 2M NaOH or 2M H₂ SO₄.Temperature was maintained at 30° C. and dissolved oxygen tension (DOT)was controlled at a value >10% air saturation by automatic control ofagitator speed. Aeration was set at 0.75 v/v/min. Oxygen utilisationrates and carbon dioxide evolution rates were determined from exhaustgas analysis performed by mass spectrometry. Product formation wasinduced by adding IPTG as a filter sterilised 1000× stock solution to afinal concentration of 200 μM when the culture OD had reached 5.

Fermentations were run with and without chloramphenicol (25 μg/ml), norequirement for antibiotic selection in the fermentation medium has beendemonstrated.

Culture supernatants were harvested 12 h post induction bycentrifugation 4200 RPM max 240 mm (1-2 l fermentations) or bytangential flow filtration (15 l and 150 l fermentations). Clarificationof broths was superior with tangential flow filtration (TFF).

5.1.2 Defined Medium Fermentations

Fermentations were made in medium SM6 or SM6A, glucose was used as theinitial carbon and energy source for all fermentations and was addedafter medium sterilisation to a concentration of 20 g/l. Culture pH wasbrought to and maintained at 6.95 by the addition of 3.6M NH₄ OH, or 2MH₂ SO₄. DOT was maintained above 10% by control of agitator speed,culture temperature was maintained at 30° C. throughout thefermentation.

IPTG inductions: Cultures were induced with IPTG (final concentration200 μM) at OD 600 nm=40. Cultures induced with IPTG were fed glucose asrequired, (either in response to OUR or as predicted by an approximateyield of 1 OD/g glucose/1).

Lactose inductions: Induction of product expression was also obtained byswitching the carbon source to lactose from glucose. Glucose was fed tosupport the culture to an OD of approximately 30 (an accumulativeaddition of 30 g/l). Lactose feeding was started at an OD ofapproximately 25, as with glucose, lactose was then fed (normally asindividual shots of 50% lactose to a concentration of 10 g/l culture) asrequired. The 50% lactose solution was held in a water bath at 55° C.after autoclaving to prevent crystallisation.

Cultures induced by IPTG were harvested 20 h post induction. Culturesinduced by lactose feeding were harvested 24-30 h after the switch fromglucose to lactose utilisation.

Where casamino acids were added to defined medium fermentations theseadditions were made 3 h post induction.

5.2 Host Strain W3110 with the Dual Origin Expression Vector

5.2.1 Complex medium fermentations

These fermentations were made as described in section 5.1.1 except thatinduction of product formation was achieved by increasing the culturetemperature when the OD had reached 20. A temperature switch to 37° C.from 30° C. and holding the culture at 37° C. was used.

5.2.2 Defined medium fermentations

These fermentations were made as described in section 5.1.2 except thatglycerol was used throughout as the carbon and energy source (startingconcentration 20 g/L). Medium SM6A could only be used with citratereduced to 1 g/L total.

Fermentations run using glucose as the carbon source resulted ininduction of product expression prior to temperature switching and inthe absence of plasmid amplification.

Cultures were fed glycerol as required, again in response to the onlineOUR data or as predicted by an approximate yield of 1 OD/g glycerol/L.

Fermentations were harvested 12 h post temperature induction bycentrifugation or TFF. Where inductions did not arrest growth (normally4-6 h post temperature shift) it was not possible to maintain the DOTabove 10% air saturation, these cultures were allowed to become oxygendepleted and harvested.

6. PERIPLASMIC EXTRACTION PROCEDURES

For certain antibody fragments significant quantities of material wereretained within the cell periplasm. Product was released from theperiplasm by incubating culture pellets or concentrated cell suspensionsharvested by centrifugations or TFF respectively. Harvested cells werewashed with phosphate buffered saline (PBS) and then incubated in Trisbuffer 100 mM pH 7.2 containing 10 mM EDTA. Incubations were at 30° C.for 4 h. Repeated incubations of the cells produced further material.

EXAMPLE C Fermentation Process for Expression of Antibody Fragments inE. coli W3110 Using Defined Medium and the Dual Origin Vector

Storage of Strains

As frozen cell suspensions (-70° C. and original OD 600 nm=2) in LB ampmedium using 12.5% glycerol as a cryoprotectant.

Preparation of Inocula

Inocula were prepared from glycerol stock cultures. 200 ml aliquots ofLB+amp (25 mg/l) were dispensed into sterile 1 l Erlenmeyer flasks andinoculated with 500 μl glycerol stock culture. These flasks wereincubated at 30° C. and 250 RPM in an orbital incubator for approx 10 hor until an OD of 3 had been obtained. Fermentors were inoculated withLB cultures at 5% culture volume.

Fermentation Conditions

Culture medium SM6 chemically defined medium. No antibiotics were addedto the SM6 medium other than carry over from the inoculum.

Carbon source: glycerol, initial concentration 20 g/l. When the cultureOD reached 15, glycerol was fed as a 50% (w/w) solution in H₂ O at arate of approx 5 g (glycerol)/l/h. This glycerol feed can alternativelybe applied as a series of batch additions to maintain glycerolsufficiency. The glycerol requirement varies with varying growthresponse to induction.

Culture temperature was controlled at 30° C.+/-0.5° C. until the culturewas induced at OD 10, 20 or 35 by switching the temperature to 37° C.where the temperature was maintained until harvest. The optimumtemperature induction profile may vary significantly with differentheterologous proteins.

Culture pH was controlled to 7.0+/-0.1 by the addition of 4M NH₄ OH or2M H₂ SO₄.

Culture dissolved oxygen tension (DOT) was maintained at a value greaterthan 20% air saturation by regulations of the stirrer speed. Aerationwas set at 0.75 v/v/m.

Cultures were harvested approx 8 h post induction.

Broths were clarified either by centrifugation (for volumes <2 l) or bytangential flow filtration (TFF) using a millipore prostack.

The invention is described above by way of example only, and variousmodifications will be apparent to those skilled in the art which fallwithin the scope of the appended claims.

We claim:
 1. A method for the production of a heterologous protein in abacterial host cell, comprising culturing a bacterial host celltransformed with an expression vector comprising a transcription unitencoding the heterologous protein, in order to express the heterologousprotein, wherein said expression vector is controlled by a single originof replication which maintains a vector copy number between 6 and 50,the transcription unit comprises a regulatable promoter and atranscriptional terminator, and the host cell is cultured in a definedmedium in the absence of antibiotic selection.
 2. A method according toclaim 1, wherein said expression vector shows structural andsegregational stability.
 3. A method according to claim 1, wherein saidtranscription unit codes for an antibody molecule.
 4. A method accordingto claim 3, wherein said transcription unit codes for (a) both anantibody heavy chain and an antibody light chain, or (b) both anantigen-binding fragment of an antibody heavy chain and anantigen-binding fragment of an antibody light chain.
 5. A methodaccording to claim 3, wherein said antibody is a humanised orCDR-grafted antibody molecule.
 6. A method according to claim 4, whereinsaid transcription unit codes for a Fab or Fab' antibody fragment.
 7. Amethod according to claim 1, wherein said expression vector furthercomprises a gene encoding a repressor which acts on said regulatablepromoter to prevent expression of said transcription unit.
 8. A methodaccording to claim 1, wherein said transcription unit is fused to a DNAsequence encoding a secretion sequence.
 9. A method according to claim1, wherein said host cell is an E. coli cell.
 10. A method according toclaim 4, wherein said antibody is a humanised or CDR-grafted antibodymolecule.
 11. A method according to claim 5, wherein said transcriptionunit codes for a Fab or Fab' antibody fragment.